Part No: P052Issued year: 2013File size: 0.48mbFile type: pdf
Pain management therapy warrants constant monitoring of therapeutic levels of prescribed drug levels in patient urine samples. The number of samples being submitted for analysis has increased dramatically in the last 10 years with improvements in high throughput automated screening capabilities. Patient samples analysis is complicated by the need for an effective sample preparation methodology that can extract target analytes from complex matrices with good efficiency. Further complicating the process is the need to enzymatically hydrolyse the glucoronidated metabolites prior to extraction from the urine matrix. A fully automated sample preparation process using a TECAN Freedom EVO® 100 was designed to incorporate both the enzymatic hydrolysis and subsequent sample preparation assay as one continuous workflow. Supported Liquid Extraction (ISOLUTE SLE+) which offers an efficient alternative to traditional liquid-liquid extraction (LLE) and solid phase extraction (SPE) techniques was used to extract a suite of pain management drugs from spiked urine samples. A recovery and quantitation assay was run on the TECAN Freedom EVO® 100 using mock patient samples to demonstrate utility of automation process.
MSACL, Pain Management, Biotage, SPE, SLE, LLE, Supported Liquid Extraction, Drugs, MSACL, San Diego, 2013
Part No: P144Issued year: 2016File size: 0.6mbFile type: pdf
The ability to extract a broad range of different drugs from a biological matrix allows for the expedited analysis of a patient sample using LC-MS/MS. Typically small molecules are extracted from matrices like urine based on their polarities. A fast and reliable sample preparation method that could be implemented to extract drugs of different polarities from urine could be used as a screening tool to quickly identify the presence of illicit drugs in patient samples using LC-MS-MS.
This poster demonstrates the utility of supported liquid extraction for the extraction of over 30 different acidic, basic and neutral drugs in urine prior to LC-MS/MS.
Part No: AN886Issued year: 2017File size: 1mbFile type: pdf
This application note describes the extraction of 96 licit and illicit drugs of abuse from urine prior to UPLC-MS/MS analysis using EVOLUTE® HYDRO CX 96-well plates.
EVOLUTE® HYDRO CX plates offer an efficient way to perform hydrolysis in the well of the extraction plate. This method provides high analyte recovery, reduced extraction time due to the elimination of a sample transfer step as well as the elimination of the column conditioning and equilibration steps, and a reduced risk for sample carryover or cross-contamination due to the elimination of the sample transfer step.
Part No: P164Issued year: 2017File size: 0.69mbFile type: pdf
Most drugs, both licit and illicit, are excreted in urine as glucuronide conjugates. Hydrolysis using beta-glucuronidase converts the glucuronidated metabolites back to their “free” or non-conjugated
form, increasing sensitivity for LC-MS/MS analysis. Hydrolysis using red abalone, abalone, and recombinant beta-glucuronidase enzymes was evaluated using an in-well hydrolysis plate to determine which provided the most efficient hydrolysis of glucuronide metabolites without affecting overall recovery of nonconjugated compounds. A glucuronide control containing 8 glucuronide compounds was used to determine the extent of hydrolysis that occurred. A non-conjugated control containing 96 licit and illicit drugs of abuse was evaluated to determine if signal suppression occurred as a result of enzyme hydrolysis.
Part No: AN893Issued year: 2018File size: 0.91mbFile type: pdf
This application note describes the fully automated extraction of carboxy-THC from urine, following base hydrolysis prior to GC/MS analysis. The method was automated using Biotage® ExtraheraTM, configured for use with EVOLUTE EXPRESS ABN columns.
The method delivers clean extracts and analyte recovery of 90% with RSD lower than 10% for carboxy-THC and its deuterated internal standard. Using the Biotage® ExtraheraTM, 24 samples were extracted in approximately 38 minutes. The limit of quantitation of 2 ng/mL is below the SAMHSA/ EWDTS confirmation cut off of 15 ng/mL for workplace testing applications.
Part No: AN892Issued year: 2018File size: 0.87mbFile type: pdf
This application note describes the fully automated extraction of carboxy-THC from urine, following base hydrolysis prior to GC/MS analysis. The method was automated using Biotage® ExtraheraTM, configured for use with ISOLUTE SLE+ columns.
The simple sample preparation procedure delivers clean extracts and analyte recoveries greater than 80% with RSDs lower than 10% for carboxy-THC and its deuterated internal standard. Using Biotage Extrahera, 24 samples are extracted in approximately 35 minutes. Limit of quantitation is below the SAMHSA/EWDTS confirmation cut off of 15 ng/mL for workplace testing applications.
Part No: P175Issued year: 2018File size: 2.14mbFile type: pdf
This poster presents a method for detection of approximately 100 drugs and metabolites in urine using mixed-mode polymeric cation exchange solid phase extraction (SPE).
Four different glucuronidase enzymes and several incubation times and temperatures were evaluated to determine optimum hydrolysis conditions for seven commonly detected glucuronide metabolites. Samples were subjected to offline hydrolysis and SPE (EVOLUTE® EXPRESS CX, Biotage, Charlotte, NC) and results compared to an SPE plate that incorporates in-well hydrolysis (EVOLUTE HYDRO CX, Biotage).
MSACL 2018, Palm Springs, CA
Part No: PPS443.V.1Issued year: 2019File size: 2.98mbFile type: pdf
Analysis of drug panels in urine samples can be challenging, and the trend towards larger panels including multiple drug classes compounds the issues faced during method development.
This white paper examines a number of aspects of sample preparation, and their impact on the success of subsequent LC-MS/MS analysis of broad urine panels.
Section 1 examines the applicability of various sample preparation techniques: supported liquid extraction, reverse phase SPE and mixed-mode SPE, to the various classes of drugs extracted. In addition, hydrolysis approaches: enzyme type and protocol used (time, temperature), are compared.
Mixed-mode reverse phase/cation exchange SPE is widely used for extraction of basic drug classes from urine, but the inclusion of drugs and metabolites that exhibit ‘non-typical’ functionality within urine panels can be problematic. Section 2 examines the impact of various parameters (interference wash strength, elution solvent composition) on analyte retention, elution and extract cleanliness with particular focus on zwitterionic (gabapentin, pregabalin) and non-ionic (carisoprodol, meprobamate) drugs.
Part No: P157Issued year: 2017File size: 0.8mbFile type: pdf
This poster demonstrates that a large urine panel, comprised of 43 DOAs, from multiple drug classes, can be simultaneously screened by mixed-mode cation exchange SPE (using EVOLUTE EXPRESS CX 96 well plates) despite their disparate intermolecular traits, by thoughtfully selecting appropriate organic wash and elution conditions that simultaneously enable sample isolation and detection along with minimizing sample matrix effects.
The extraction method is automated using the Biotage® Extrahera™ Automated sample Preparation Platform.
MSACL 2017, Palm Springs
SOFT 2017, Boca Raton
Part No: P156Issued year: 2017File size: 0.23mbFile type: pdf
Most drugs are excreted in urine as glucuronide conjugates. Hydrolysis using a beta-glucuronidase enzyme to convert the metabolites to their “free” form for analysis increases sensitivity. Red abalone (Kura Biotech), abalone (Campbell Scientific), and recombinant (IMCSzyme) beta-glucuronidase enzymes were evaluated to determine which provided the most complete hydrolysis of glucuronide metabolites without effecting the overall recovery of non-conjugated compounds.
EVOLUTE EXPRESS CX 96-well plates were used to extract hydrolysed urine samples, and the impact of th enzymes was compared.
MSACL 2017, Palm Springs
SOFT 2017, Boca Raton
Part No: AN841Issued year: 2015File size: 0.82mbFile type: pdf
This application note describes the extraction of 11-nor-9-carboxy-THC from a urine matrix, prior to GC/MS analysis.
Carboxy-THC is the primary metabolite of THC, a key indicator of illicit marijuana usage. In urine, ~80% of the carboxy-THC metabolite is present in the form of its glucuronide metabolite. Therefore, to effectively quantify the THC-COOH, urine is hydrolyzed before extraction. This application note describes optimized extraction of urine samples prepared by either enzymatic or base hydrolysis.
Part No: P050Issued year: 2013File size: 0.46mbFile type: pdf
The use of schedule I drugs for patient pain management therapy warrants constant monitoring of therapeutic levels in patient urine samples. Screening patient urine samples is further complicated by the metabolic process which converts the free drug to the - glucuronide form. The target drugs in patient urine samples can be enzymatically hydrolysed and extracted using Supported Liquid Extraction (ISOLUTE SLE+) which offers an efficient alternative to traditional liquid-liquid extraction (LLE) and solid phase extraction (SPE) techniques typically used for this type of clinical sample preparation.
San Diego, MSACL, 2013
Part No: AN768Issued year: 2013File size: 1.86mbFile type: pdf
This application note describes a Supported Liquid Extraction (SLE) protocol for the extraction of various drugs of abuse from hydrolyzed urine prior to LC-MS/MS analysis.
drugs of abuse, sle, multiclass
Part No: AN771Issued year: 2012File size: 0.41mbFile type: pdf
This application note describes effective and efficient ISOLUTE SLE+ protocols optimized for sample (hydrolysed urine) volumes of either 200 μL or 1 mL. Procedures for high throughput (96-well plate) and column format are included. The simple sample preparation procedure delivers clean extracts and analyte recoveries of 82-100% with RSDs of <5% for all analytes. Sub ng/mL LOQs can be achieved.
Part No: AN766Issued year: 2013File size: 1.26mbFile type: pdf
This application note describes effective and efficient protocols for extraction of cocaine and its major metabolites optimized for sample loading volumes of either 400 μL or 1 mL using ISOLUTE SLE+ supported liquid extraction columns. The simple sample preparation procedure delivers clean extracts and analyte recoveries greater than 83% with RSDs of <7% for all analytes.
Part No: AN915Issued year: 2019File size: 1.53mbFile type: pdf
This application note describes the extraction of 25 illicit and prescribed drugs from hydrolyzed urine using ISOLUTE® HYDRO DME+ Dual Mode Extraction plates prior to UPLC-MS/MS analysis.
Because of the enhanced sample clean up delivered by ISOLUTE HYDRO DME+ products, analyte sensitivity is significantly increased compared to traditional dilute and shoot (D&S) techniques, resulting in reduced limits of quantitation (LOQ)
Part No: AN780Issued year: 2013File size: 1.33mbFile type: pdf
This application note describes effective and efficient protocols optimized for sample loading volumes of either 400 μL or 1 mL using ISOLUTE SLE+ supported liquid extraction columns. The simple sample preparation procedure delivers clean extracts and analyte recoveries greater than 83% with RSDs of <10% for all analytes.
Part No: P145Issued year: 2016File size: 0.42mbFile type: pdf
This poster demonstrates a rapid and reliable sample preparation method using Supported Liquid Extraction (ISOLUTE SLE+) to extract a suite of 30 hormone analytes from a hydrolyzed urine matrix. Single injection analysis by LC-MS/MS shows that matrix effects are eliminated by the ISOLUTE SLE+ protocol and that analyte recovery and sensitivity have excellent clinical utility.